The present invention relates to the preparation and use of vesicles and related encapsulating membranes made in aqueous solution from amphiphilic polymers and related molecules.
Membranes that are stable in aqueous media are heavily relied upon for compartmentalization by biological cells. For instance, the outermost plasma membrane of a cell separates the inside of a cell from the outside and, like most cell membranes, it is a self-assembled, complex fluid of biological molecules, primarily lipids and proteins. Only a few molecules, such as water and small, uncharged organic molecules, significantly permeate the membrane. A biomembrane also possesses stability and other thermo-mechanical properties that are not unrelated to passive permeability and are certainly central to cell function (see, e.g. Lipowsky and Sackmann, Eds., Structure and Dynamics of Membranes from Cells to Vesicles, Handbook of Biological Physics. vol 1 (Elsevier Science, Amsterdam, 1995); Bloom et al., Q. Rev. Biophys. 24:293 (1991)).
The same characteristics of permeability and thermo-mechanical stability in addition to biocompatibilityxe2x80x94also affect how lipid vesicles that are assembled in vitro and that are also known as liposomes can effectively encapsulate and deliver a long list of bioactive agents (Needham et al., in Vesicles, M. Rosoff, Ed. (Dekker, N.Y., 1996), chap. 9; Cevc and Lasic in Handbook of Biological Physics, chaps. 9-10, 1995; Koltover et al., Science 281:78 (1998); Harasym et al., Cancer Chemother. Pharmacol. 40:309 (1997)). The typical liposome is comprised of one or more bilayer membranes, each approximately 5 nm thick and composed of amphiphiles such as phospholipids. Each bilayer exists as a temperature- and solvent-dependent lamellar phase that is, in its surface, in a liquid, gel, or liquid-gel coexisting state. Because of a certain intrinsic biocompatibility of phospholipid vesicles, many groups have developed them for use as encapsulators and delivery vehicles. Vesicles surrounded by a lipid bilayer can range in diameter from as small as tens of nanometers to giants of 0.5-40 microns.
Phospholipid vesicles are materially weak and environmentally sensitive. Transit through the digestive tract, for example, can expose liposomes to a host of solubilizing agents. Repeated transit through the microcirculation can also tear apart giant phospholipid vesicles which cannot withstand high fluid shear. Smaller phospholipid vesicles may not fragment, but they tend to adhere, and are thus cleared from circulation. Circulating cells suppress their own adhesion partly through a brushy biopolymer layer, known as the glycocalyx, which faces the environment. The glycocalyx has, to some extent, been mimicked in liposome systems by the covalent addition to lipids of hydrophilic polyethyleneglycol (PEG) polymer chains. To maximally extend a vesicle""s circulation lifetime (about ten hours), a suitable PEG weight ranges between about two and five kilograms/mole.
To further counteract mechanical forces imposed on their membranes, cells often also possess a sub-membranous network of cross-linked proteins (Alberts et al., in Molecular Biology of the Cell, 3rd ed., pp489-493 and 800-1, Garland Publ., Inc., New York, 1999). The red blood cell, as an example, survives repeated deformation through the microcirculation without fragmentation, but only because it has a cross-linked network of peripheral membrane proteins. Without such a network, the cells cannot withstand such circulation for more than a few hours even with a glycocalyx (Schmid-Schoenbein et al., Blut 52(3):131 (1986)). With a normal membrane network, red blood cells circulate in humans for more than 100 days. In terms of measurable properties, the network imparts a shear elasticity that is only achievable with a cross-linked structure.
Past efforts to enhance the stability of lipid lamellae against shear and other factors, resulted in the synthesis of many different modified lipid molecules with polymerizable double bonds. Such bonds were located either at the surfactant head group, or more, commonly, at different locations on the hydrophobic tails (Fendler et al., Science 223:888 (1984); Liu et al, Macromolecules 32:5519 (1996)). This approach clearly had the ability to generate covalently inter-connected poly-amphiphiles when reacted after self-assembly into membranes per ordinary lipids. However, a fully, covalently interconnected network of lipids requires complete cross-linking of the membrane of a vesicle, and the full extent of cross-linking achievable with cross-linkable lipids appears to be difficult to ascertain. O""Brien""s group (Sisson et al., Macromolecules 29:8321 (1996)) has used solubility in hexafluoropropanol to estimate a degree of polymerization up to at least 1000. This corresponds to a vesicle diameter of about 10 nanometers, if one assumes complete cross-linking within and between layers of the bilayer, and a typical lipid area of about 0.5 square nanometers per lipid. Detergent induced leakage of entrapped solutes was strongly inhibited by cross-linking. It is clear, however, that no fully cross-linked lipid vesicle larger than several hundred nanometers has been reported.
A cross-liinkable amphiphile related to cross-linkable phospholipids has been made by Komatsu et al., J. Am. Chem. Soc. 119:11660 (1997)). Tetrakis(aminophenyl)porphyrin contains four hydrophobic bixin side chains that each terminate in a small hydrophilic carboxylate group and harbor approximately ten (photo)reactive double bonds along the backbone of each bixin chain. When dissolved in the organic solvent, tetrahydrofuran (THF), and rapidly injected into a one-eighth volume of water and sonicated, the synthetic molecules reportedly formed vesicles. However, the resulting membranes are porous. Irradiation led to what was claimed to be the first spherical membrane structure of molecular thickness, which was considered a single, dehydratable, balloon-shaped polymer molecule insoluble in a predominantly organic solvent, such as 95% ethanol. Electron micrographs showed spherical particles of less than 100 nm, while collapsed particles studied by atomic force microscopy were reported to have a height of about 7 nm. Whether the cross-linked shells were truly semi-permeable vesicles or were highly porous macromolecular shells, as Komatsu et al. suggested, leaves open the question of whether, to date, a wholly cross-linked vesicle of any size has actually been produced. Certainly, no cross-linked vesicle larger than several hundred nanometers has been reported.
Small amphiphiles of natural origin, such as phospholipids have inspired the engineering of high molecular weight analogs, which also self-assemble into complex phases in aqueous media similar to those observed for phospholipids. For example, vesicles have been assembled in aqueous solution by Uchegbu et al., J. Pharm. and Pharmacol. 50:453 (1998)) using the naturally occurring macromolecule chitosan modified by the covalent attachment of many fatty acid pendant groups. The resulting self-assembled vesicles were 300-600 nanometers in diameter, and were shown to be bio- and haemocompatible. Although such modified natural products have disadvantages of variability in the natural polymer and a lack of precise control in covalent modification, the assembly of membranes from amphiphiles of high molecular weight has the potential to improve vesicle stability. The overall approach has similarities to lipid cross-linking, but a primary distinction lies in the fact that, with cross-linking, self-assembly of the membrane must occur first.
Many semi- or partially-synthetic, amphiphilic molecules are also significantly larger (in molecular weight, volume, and linear dimension) than phospholipid amphiphiles, and have therefore been called xe2x80x9csuper-amphiphilesxe2x80x9d (Comelissen et al., Science 280:1427 (1998)). Cornelissen et al. used polystyrene (PS) as a hydrophobic fraction in their series of non-synthetic, natural block copolymers designated PS40-b-(isocyano-L-alanine-L-alanine)y. For y=10, but not y=20 or 30, loop structures, referred to as small collapsed vesicles, having diameters ranging from tens of nanometers to several hundred, and a bilayer thickness of 16 nanometer were mentioned as existing under a single acidic buffer condition (0.2 mM Na-acetate buffer, pH 5.6). However, bilayer filaments and superhelical rods existed, without explanation, under the same solution conditions, thus making the stability of the collapsed vesicles, relative to the other microstructures, highly uncertain for the studied dipeptide-based copolymer. Furthermore, no demonstration of semi-permeability was reported, and reasons for apparent vesicle collapse were not given, further raising questions of vesicle stability.
Additional spherical shell structures smaller than a few hundred nanometers, and which required the presence of organic solvents mixed into water to drive their formation, include those assembled from various block copolymers as observed by Yu et al., Macromolecules 31:1144 (1998); Ding et al., J. Phys. Chem. B 102:6107 (1998); Henselwood et al., Macromolecules 31:4213 (1998)). However, there appears in the prior art only one example of a wholly synthetic super-amphiphile that has the unpredicted capacity to self-assemble in aqueous solution, albeit only under moderately acidic pH conditions, into a vesicle-like microstructure, and that is the reported work of Cornelissen et al., 1998, although even those structures were of questionable state and stability.
Both amphiphiles and super-amphiphiles can exist in a broad variety of microphases and bulk phases that include not only lamellar, but also hexagonal, cubic, and more exotic phases (see review by Lipowsky and Sackmann, in Handbook of Biological Physics, 1995; Bates, Science 251:898 (1991). Based on the work of Hajduk et al. (see, J. Phys. Chem. B 102:4269 (1998)), the ability of super-amphiphilic block copolymers to form lamellar phases in aqueous solutions can be regulated by both synthetic tuning of polymer chemistry and physical variables, such as concentration and temperature. Evidence has now accumulated that in dilute solutions certain diblock copolymers, such as polyethyleneoxide-polyethylethylene (PEO-PEE, wherein PEO is structural equivalent to PEG), can form not only worm-like inicelles (Won et al., Science 283:960-3 (1999)), but also unilamellar vesicles (Discher et al., Science 284:1143 (1999)).
In addition, because of the synthetic control over molecular composition, properties of membranes assembled from super-amphiphiles can be controlled in novel ways. For instance, a super-amphiphilic polymer can be made far more reactive than a much smaller phospholipid molecule simply because more reactive groups can be designed into the polymer. The principle was first illustrated for the aforementioned worm-like micelles in which polyethyleneoxide-polybutadiene (PEO-PBD) mesophases were successfully cross-linked into bulk materials with completely different properties, notably an enhanced shear elasticity (Won et al., 1999). The resulting microstructures, though assembled in water, could withstand dehydration, as well as exposure to an organic solvent, such as chloroform. In the absence of cross-linking, microstructures of amphiphiles and super-amphiphiles aregenerally unstable to treatments that could otherwise prove very useful for a range of applications that might benefit from, for example, sterilization, or long-term dry storage.
Despite recent advances, there remained until the present invention a long felt need in the art for stable, aqueous-formed vesicles which could be more broadly engineered but still have demonstrable features in common with a biomembrane or a mimic, including: biocompatibility, selective permeability to solutes, the ability to retain internal aqueous components and control their release, the ability to deform yet be relatively tough and resilient, and the ability to extensively cross-link within the membrane in order to withstand extreme environments.
The present invention meets the need in the art by providing not only an illustrative set of stable super-amphiphilic vesicles in biocompatible, aqueous solutions, but it also provides vesicles which are entirely synthetic, creating an opportunity to tailor the dynamics, structure, rheological and even optical responses of the membrane based on its composition. The polymer vesicles of the present invention are called xe2x80x9cpolymersomes.xe2x80x9d Analogous to xe2x80x9cliposomesxe2x80x9d made from phospholipids, the material properties of the polymersome vesicles can be readily measured using techniques that have been largely developed for phospholipid vesicles and biological cells. Furthermore, the ability to cross-link the polymer building blocks affords a novel opportunity to provide mechanical control and stability to the vesicle on the order of that which is provided by the protein skeleton at a cell""s plasma membrane.
Polymersomes of the present invention possess membranes capable of self-repair, adaptability, portability, resilience, and are selectively permeable, thereby providing, for example, long-term, reliable and controllable vehicles for the delivery or storage of drugs or other compositions, such as oxygen, to the patient via the bloodstream, gastrointestinal tract, or other tissues, as replacement artificial tissue or soft biomaterial, as optical sensors, and as a structural basis for metal or alloy coatings to provide materials having unique electric or magnetic properties for use in high-dielectric or magnetic applications or as microcathodes.
In accordance with the present invention, there are provided vesicles comprising semi-permeable, thin-walled encapsulating membranes, wherein the membranes are formed in an aqueous solution, and wherein the membranes comprise one or more synthetic super-amphiphilic molecules. The invention relates to all super-amphiphilic molecules, which have hydrophilic block fractions within the range of 20-50% by weight, and which achieve some or all of the above capsular states of matter. Further provided are vesicles and encapsulating membranes, wherein at least one super-amphiphile molecule is a block copolymer, and wherein the resulting vesicle is termed a polymersome. The thus provided polymersomes may be comprised of multi-block copolymers, most preferably, but not limited to diblock or triblock copolymers. Moreover, in certain preferred embodiments of the present invention are provided polymersomes in which all of the super-amphiphile molecules are block copolymers. The block copolymers useful in the present invention may be selected from any known block copolymer, including, for example polyethylene oxide (PEO), poly(ethylethylene) (PEE), poly(butadiene) (PB or PBD), poly(styrene) (PS), and poly(isoprene) (PI). As needed, monomers for these polymers will be denoted by EO, EE, B or BD, S, and I, respectively.
In addition the present invention provides polymersomes, wherein the vesicles are capable of self-assembly in aqueous solution.
The present invention also provides methods for the preparation of mixtures of super-amphiphiles from smaller amphiphiles, such as phospholipids up to at least 20% mole fraction, which have also been shown capable of integrating into stable encapsulating membranes.
Further provided in the present invention are reactive amphiphiles that can be covalently cross-linked together, over a many micron-squared surface, while maintaining semi-permeability of the membrane. Cross-linked polymersome are characterized as having the ability to withstand exposure to organic solvents, boiling water, dehydration and rehydration in an aqueous solution without visibly or significantly affecting the integrity of the membrane.
In addition, the present invention provides polymersomes, wherein the vesicle is biocompatible. Further provided are vesicles for the retention, delivery, and/or extraction of materials, which may require membrane biocompatibility and may or may not take advantage of the novel thermal, mechanical, or chemical properties of the surrounding membranes.
The present invention also provides polymersomes which encapsulate one or more compositions, such as a drug, therapeutic compound, dye, nutrient, sugar, vitamin, protein or protein fragment, salt, electrolyte, gene or gene fragment, product of genetic engineering, steroid, adjuvant, biosealant, gas, ferrofluid, or liquid crystal. The polymersome may be further used to transport an encapsulatable material to or from its immediately surrounding environment.
Moreover, the present invention provides methods of using the polymersome or encapsulating membrane to transport one or more of the above identified compositions to or from a patient in need of such transport activity. For example, the polymersome could be used to deliver a drug or therapeutic composition to a patient""s tissue or blood stream, or it could be used to remove a toxic composition from the blood stream of a patient with, for example, a life threatening hormone or enzyme imbalance.
Also provided by the present invention are methods of preparing a polymersome, wherein the preferred methods of preparation include at least one step consisting of a film rehydrating step, a bulk rehydrating step, or an electroforming step.
Further provided are methods for controlling the release of an encapsulated material from a polymersome by modulating and controlling the composition of the membrane. For example, one preferred method of controlling the release of an encapsulated material from a polymersome or encapsulating membrane entails cross-linking the membrane. In another preferred method, release of the encapsulated material is controlled by forming the encapsulating membrane from at least one cross-linkable amphiphile and at least one non cross-linkable molecule, followed by subjecting the thus destabilized membrane to chemical exposure or to waves of propagated light, sound, heat, or motion.
In addition, the present invention provides an encapsulating membrane comprising a semi-permeable, thin-walled encapsulating, amphiphilic membrane, wherein the membrane is formed around a droplet of oil in a microemulsion of oil dispersed in an aqueous solution, and wherein the membrane comprises one or more synthetic super-amphiphilic molecules. In a preferred embodiment, a super-amphiphile layer self-assembles around the oil droplet in water, with or without cross-linking of the super-amphiphile.
These and other aspects of the invention will become more apparent from the following detailed description when taken in conjunction with the following figures.